EGFR Mutant Autophosphorylation Assay

The pEGFP-N1-EGFR plasmid from our previous studies^{15 (link),17 (link)} was used to generate point mutations. Before transfection, Chinese Hamster Ovary (CHO) cells were grown in high-glucose Dubecco’s Modified Eagle Medium (DMEM) (Cellgro, Manassas, VA, USA) with 10% fetal bovine serum (Bioexpress, UT, USA) without antibiotics at 30% confluency. Transient transfection was then performed using lipofectamine-2000 (Invitrogen, Carlsbad, CA) according to manufacturer’s protocol with WT and mutant EGFR. To detect autophosphorylation, transfected cells were serum-starved in Ham’s F-12 media for 18 h followed by ligand stimulation with EGF (100 ng/mL) (Sigma, St. Louis, MO) for 5 min. Cells were washed with PBS, and lysed with lysis buffer (50 mM Tris-HCL, pH 7.4, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM sodium orthovanadate, 1% Triton X-100, 1 mM PMSF, and 1× Protease Inhibitor Cocktail Set V, EDTA-free). Total protein was resolved on 10% SDS-PAGE and transferred onto polyvinylidenedifluoride (PVDF) membrane. Western blotting was done using anti-GFP, anti-pY1197-EGFR, anti-FLAG, anti-STAT3, and anti-pY705-STAT3 antibodies (Cell signaling, Danvers, MA). Proteins were detected using chemiluminescent substrate (ECL substrate, Pierce, Rockford, IL).